Robustness of hen lysozyme monitored by random mutations

Area of Science:

• Protein Engineering
• Biochemistry
• Molecular Biology

Background:

• Hen lysozyme is a model protein for studying protein stability.
• Understanding protein robustness is crucial for protein design and function.

Purpose of the Study:

• To investigate the impact of random mutations on hen lysozyme's structure and activity.
• To determine the robustness of hen lysozyme's gross conformation and active structure.

Main Methods:

• Construction of six random mutant libraries of hen lysozyme with varying mutation levels (1-14 amino acids).
• Systematic variation of Mg(2+) and Mn(2+) concentrations during polymerase chain reaction.
• Screening of 4000 clones using lysis activity assays and ELISA with a conformation-specific monoclonal antibody.

Main Results:

• Approximately 80% of clones with an average of two mutations retained active structure.
• Nearly all clones with an average of five mutations lost active structure.
• 80% of clones with an average of two mutations maintained gross conformation, while 24% with 14 mutations retained gross conformation.

Conclusions:

• Hen lysozyme's gross conformation is highly robust against mutations.
• The active structure of hen lysozyme is also robust, but to a lesser extent than its gross conformation.